On the mechanism for efficient repression of the interleukin-6 promoter by glucocorticoids: enhancer, TATA box, and RNA start site (Inr motif) occlusion.

The feedback inhibition of interleukin-6 (IL-6) gene expression by glucocorticoids represents a regulatory link between the endocrine and immune systems. The mechanism of the efficient repression of the IL-6 promoter by dexamethasone (Dex) was investigated in HeLa cells transiently transfected with plasmid constructs containing different IL-6 promoter elements linked to the herpesvirus thymidine kinase gene (tk) promoter and the bacterial chloramphenicol acetyltransferase gene (cat) and cotransfected with cDNA vectors constitutively expressing either the active wild-type or inactive mutant human glucocorticoid receptor (GR). The induction by interleukin-1, tumor necrosis factor, phorbol ester, or forskolin of IL-6-tk-cat chimeric constructs containing a single copy of the IL-6 DNA segment from -173 to -151 (MRE I) or from -158 to -145 (MRE II), which derive from within the multiple cytokine- and second-messenger-responsive enhancer (MRE) region, was strongly repressed by Dex in a wild-type GR-dependent fashion irrespective of the inducer used. The induction by pseudorabies virus of an IL-6 construct containing the IL-6 TATA box and the RNA start site ("initiator" or Inr element) but not the MRE region was also repressed by Dex in the presence of wild-type GR. DNase I footprinting showed that the purified DNA-binding fragment of GR bound across the MRE, the TATA box, and the Inr site in the IL-6 promoter; this footprint overlapped that produced by proteins present in nuclear extracts from uninduced or induced HeLa cells. Imperfect palindromic nucleotide sequence motifs moderately related to the consensus GR-responsive element (GRE) motif were present at the Inr, the TATA box, and the MRE II site in the IL-6 promoter; although MRE I and a GR-binding site between -201 and -210 in IL-6 both lacked a discernible inverted repeat motif, their sequences showed considerable similarity with negative GRE sequences in other Dex-repressed genes. Surprisingly, chimeric genes containing MRE II, which lacks a recognizable GACGTCA cyclic AMP- and phorbol ester-responsive motif, were strongly induced by both phorbol ester and forskolin, suggesting that MRE II (ACATTGCACAATCT) may be the prototype of a novel cyclic AMP- and phorbol ester-responsive element. Taken together, these observations suggest that ligand-activated GR represses the IL-6 gene by occlusion not only of the inducible IL-6 MRE enhancer region but also of the basal IL-6 promoter elements.     

Synthesis of interleukin 6 (interferon-beta 2/B cell stimulatory factor 2) in human fibroblasts is triggered by an increase in intracellular cyclic AMP

Interleukin 6 (IL-6; also referred to as interferon-beta 2, 26-kDa protein, and B cell stimulatory factor 2) is a cytokine whose actions include a stimulation of immunoglobulin synthesis, enhancement of B cell growth, and modulation of acute phase protein synthesis by hepatocytes. Synthesis of IL-6 is stimulated by interleukin 1 (IL-1), tumor necrosis factor (TNF), or platelet-derived growth factor. We examined the role of the cyclic AMP (cAMP)-dependent signal transduction pathway in IL-6 gene expression. Several activators of adenylate cyclase, including prostaglandin E1, forskolin, and cholera toxin, as well as the phosphodiesterase inhibitor isobutylmethylxanthine and the cAMP analog dibutyryl cAMP, shared the ability to cause a dramatic and sustained increase in IL-6 mRNA levels in human FS-4 fibroblasts. Actinomycin D treatment abolished this enhancement. Treatments that increased intracellular cAMP also stimulated the secretion of the IL-6 protein in a biologically active form. Increased intracellular cAMP appears to enhance IL-6 gene expression by a protein kinase C-independent mechanism because down-regulation of protein kinase C by a chronic exposure of cells to a high dose of 12-O-tetradecanoylphorbol 13-acetate did not abolish the enhancement of IL-6 expression by treatments that increase cAMP. IL-1 and TNF too increased IL-6 mRNA levels by a protein kinase C-independent mechanism. Our results suggest a role for the cAMP-dependent pathway(s) in IL-6 gene activation by TNF and IL-1.     

Enhanced phosphorylation of 65 and 74 kDa proteins by tumor necrosis factor and interleukin-1 in human peripheral blood mononuclear cells

Tumor necrosis factor (TNF) and interleukin-1 (IL-1) enhanced the phosphorylation of identical cytosolic 65 kDa protein (P65 or l-plastin) and 74 kDa protein (P74) at serine residues in human peripheral blood mononuclear cells (PBMC). The isoelectric points of P65 and P74 were 5.6 and 4.7 to 5.0, respectively. The phosphorylation of these proteins increased with a few minutes and reached maximal levels of approximately 3 times the unstimulated levels by 10 minutes. The phosphorylation of P65 and P74 was extensively enhanced by a potent protein kinase C (PKC) activator, PMA. However, there was no translocation of PKC from cytosol to membrane in PBMC that was stimulated with either TNF or IL-1, which suggests that PKC does not participate in TNF or IL-1 signal transduction. cAMP dependent protein (PKA) activators, forskolin and PGE2, failed to increase the phosphorylation, which is in agreement with the data showing that neither TNF nor IL-1 increased cAMP levels in PBMC. These results suggest that induction of phosphorylation of P65 and P74 by TNF and IL-1 is not mediated by PKC and PKA but may be mediated by another protein kinase and result in overlapping of biological activities between TNF and IL-1.     

Genetic polymorphisms within tumor necrosis factor gene promoter region: a role for susceptibility to ventilator-associated pneumonia

Debatable findings exist among various studies regarding the impact of single nucleotide polymorphisms (SNPs) within the promoter region of the tumor necrosis factor (TNF) gene for susceptibility to infections. Their impact was investigated in a cohort of mechanically ventilated patients who developed ventilator-associated pneumonia (VAP). Two-hundred and thirteen mechanically ventilated patients who developed VAP were enrolled. Genomic DNA was extracted and SNPs at the -376, -308 and -238 position of the promoter region of the TNF gene were assessed by restriction fragment length polymorphisms. Monocytes were isolated from 47 patients when they developed sepsis and stimulated by bacterial endotoxin for the production of TNFα and of interleukin-6 (IL-6). Patients were divided into two groups; 166 patients bearing only wild-type alleles of all three studied polymorphisms; and 47 patients carrying at least one A allele of the three studied SNPs. Time between start of mechanical ventilation and advent of VAP was significantly shorter in the second group than in the first group (log-rank: 4.416, p: 0.041). When VAP supervened, disease severity did not differ between groups. Stimulation of TNFα and of IL-6 was much greater by monocytes for patients carrying A alleles. Carriage of at least one A allele of the three studied SNPs at the promoter region of the TNF-gene is associated with shorter time to development of VAP but it is not associated with disease severity. Findings may be related with a role of the studied SNPs in the production of pro-inflammatory cytokines.     

Interleukin-6 repression is associated with a distinctive chromatin structure of the gene.

Expression of the interleukin-6 (IL-6) gene is usually tightly controlled and may be induced in specific tissues only after treatment with appropriate stimuli. The molecular mechanisms responsible for IL-6 gene repression in specific tissues or cell lines remain poorly defined. In order to address this question we have studied two human breast carcinoma cell lines, MDA-MB-231, in which the IL-6 gene is expressed, and MCF-7, in which it is not. The promoter region of the IL-6 gene was analysed in both cell lines with reference to two different parameters: (i) DNase I hypersensitivity; (ii) the in vivo pattern of DNA-protein interactions. We show herein that the mechanism responsible for silencing IL-6 gene expression in MCF-7 cells most probably involves a modification of chromatin structure, as suggested by a decreased sensitivity of the IL-6 promoter to DNase I relative to the IL-6-expressing cell line MDA-MB-231. Moreover, we show that a 'closed' nucleosomal structure in MCF-7 cells does not inhibit the binding of nuclear proteins to IL-6 gene regulatory sequences in vivo. We suggest, therefore, that, in non-expressing cells, local chromatin remodelling at the proximal promoter is inhibited by negative regulators, as suggested by two specific hallmarks of nuclear factor binding that are not observed in expressing cells: an additional in vivo footprint spanning positions -135/-119 and an additional DNase I hypersensitive site far upstream, around position -1400. Furthermore, a specific factor binding in vitro to the -140/-116 region of the IL-6 promoter is found in MCF-7 cells.     

Endogenous production of tumor necrosis factor by primary cultures of murine calvarial cells: influence on IL-6 production and osteoclast development

We have previously shown that tumor necrosis factor (TNF) and interleukin-1 (IL-1) acted synergistically to stimulate the production of IL-6 by bone marrow stromal and osteoblastic cells; and that an antibody to IL-6 inhibited TNF-induced osteoclast development in murine calvarial cell cultures. Prompted by this evidence, we have now examined whether TNF and/or IL-1 are produced by murine calvarial cells, and whether these cytokines are involved in IL-6 production and osteoclast formation. When cultured under basal conditions, calvarial cells produced TNF and IL-6, and were able to form bone resorbing osteoclasts. A neutralizing antibody against TNF suppressed both basal IL-6 production and the formation of bone resorbing osteoclasts. The anti-TNF antibody also inhibited IL-6 production in response to exogenous IL-1 or parathyroid hormone (PTH). In contrast, a neutralizing anti-IL-1 receptor antibody had no effect on basal, TNF- or PTH-stimulated IL-6 production. These findings suggest that TNF, but not IL-1, is produced by murine bone cells and that endogenous TNF induces the IL-6 production, osteoclast formation, and bone resorption exhibited by these cultures under basal conditions. Furthermore, bone cell-derived TNF amplifies the stimulatory effect of exogenous IL-1 or PTH on IL-6 production by calvarial cells.     

Interferon-regulated Mx genes are not responsive to interleukin-1, tumor necrosis factor, and other cytokines.

Accumulation of Mx gene products in cells of patients and experimental animals has been recognized as a useful marker for detecting minute quantities of biologically active interferon (IFN). Goetschy et al. (J. Goetschy, H. Zeller, J. Content, and M. A. Horisberger, J. Virol. 63:2616-2622, 1989) reported that not only IFNs but also interleukin-1 (IL-1) and tumor necrosis factor (TNF) were potent inducers of the human Mx genes. However, we observed no Mx induction in cultured human fibroblasts or in human peripheral blood mononuclear cells treated with various concentrations of IL-1 alpha or TNF-alpha. Mx induction was found in the spleens of mice treated with TNF-alpha or IL-1 alpha, but this effect could be neutralized with antibodies to murine IFN-alpha/beta. Of the other cytokines that we tested (IL-2, IL-6, and granulocyte-macrophage colony-stimulating factor), only IL-2 induced the Mx genes in peripheral blood mononuclear cells, but antibodies to human IFN-beta efficiently neutralized this effect. Our results thus indicate that IFNs are the only cytokines with intrinsic Mx-inducing activity.     

Tumor necrosis factor alpha and interleukin-1beta regulate the murine manganese superoxide dismutase gene through a complex intronic enhancer involving C/EBP-beta and NF-kappaB.

Manganese superoxide dismutase (MnSOD), a tumor necrosis factor (TNF)-inducible reactive oxygen-scavenging enzyme, protects cells from TNF-mediated apoptosis. To understand how MnSOD is regulated, transient transfections of promoter-reporter gene constructions, in vitro DNA binding assays, and in vivo genomic footprint (IVGF) analysis were carried out on the murine MnSOD gene. The results of this analysis identified a 238-bp region of intron 2 that was responsive to TNF and interleukin-1beta (IL-1). This TNF response element (TNFRE) had the properties of a traditional enhancer element that functioned in an orientation- and position-independent manner. IVGF of the TNFRE revealed TNF- and IL-1-induced factor occupancy of sites that could bind NF-kappaB and C/EBP. The 5' portion of the TNFRE bound C/EBP-beta in vitro and was both necessary and sufficient for TNF responsiveness with the MnSOD promoter or with a heterologous promoter when in an upstream position. The 3' end of the TNFRE bound both NF-kappaB and C/EBP but was not necessary for TNF responsiveness with the MnSOD promoter. However, this 3' portion of the TNFRE was required for the TNFRE to function as a downstream enhancer with a heterologous promoter. These data functionally separate the MnSOD TNFRE into a region responsible for TNF activation and one that mediates induction when it is downstream of a promoter.     

Induction of phospholipase A2 gene expression in human hepatoma cells by mediators of the acute phase response.

Serum levels of phospholipase A2 (PLA2) activity have been shown to be elevated in cases of septic shock and rheumatoid arthritis. The cellular origin of serum PLA2, however, is not known. In this report, we demonstrate that human group II PLA2 expression and secretion are induced in hepatoma cells (HepG2) following treatment with interleukin-6 (IL-6), tumor necrosis factor (TNF), and interleukin-1 (IL-1). Of the three cytokines, IL-6 is the most potent. Significant synergy is observed between IL-6 and IL-1 and between IL-6 and TNF, but not between IL-1 and TNF. PLA2 induction does not occur in human YT cells, which are known to have receptors for both IL-1 and IL-6, indicating that the regulatory mechanism involved is cell type-specific. The results of RNA blot analysis indicate that the PLA2 gene is regulated in HepG2 cells at the pretranslational level. Induction of PLA2 synthesis in HepG2 cells in response to these cytokines resembles the induction of the acute phase plasma proteins which are synthesized in cultured hepatocytes and hepatoma cells following exposure to the same cytokines and in liver in response to inflammation and infection. In addition, a putative IL-6-responsive element, which is homologous to a similar element found in several acute phase genes, is present in the 5'-promoter-proximal region of the PLA2 gene. These results suggest that serum PLA2 is synthesized in and secreted from liver cells in response to inflammatory stimuli, mediated primarily by IL-6, and therefore should be classified as an acute phase protein.     

Effects of the human immunodeficiency virus type 1 Tat protein on the expression of inflammatory cytokines.

Increased levels of inflammatory cytokines, including tumor necrosis factor (TNF), interleukin-1 (IL-1), and IL-6, have been detected in specimens from human immunodeficiency virus type 1 (HIV-1)-infected individuals. Here we demonstrate that HIV-1 activates the expression of TNF but not of IL-1 and IL-6 in acutely and chronically infected T cells. The increase in TNF gene expression is due to activation of the TNF promoter by the viral gene product Tat. Transactivation of TNF gene expression requires the product of the first exon of the tat gene and is cell type independent. T cells chronically infected with pol-defective HIV-1 provirus constitutively express both Tat and TNF at levels significantly higher (fivefold) than those seen in control cells, and treatment with phorbol myristate acetate greatly enhances Tat expression and TNF production. As TNF can increase the production of IL-1 and IL-6 and these inflammatory cytokines all enhance HIV-1 gene expression and affect the immune, vascular, and central nervous systems, the activation of TNF by Tat may be part of a complex pathway in which HIV-1 uses viral products and host factors to increase its own expression and infectivity and to induce disease.